1. Field of the Invention
The present invention relates to methods and reagents for the covalent attachment of specific binding members to a solid support. In particular, the invention relates to the immobilization of specific binding members on solid supports for use in diagnostic assays and separation procedures. The present invention is also related to the co-owned and copending patent application entitled "Method for Renaturing Proteins in the Presence of Alkyl Sulfate Detergents", filed concurrently herewith and incorporated by reference herein.
2. Description of Related Art
Binding assays have found widespread application in the field of clinical diagnostics for the detection and/or measurement of drugs, hormones, proteins, peptides, metabolites, microorganisms and other substances of interest, commonly referred to as analytes, in both biological and non-biological fluids. Binding assays incorporate specific binding members, typified by antibody and antigen immunoreactants, wherein one member of the specific binding pair is labeled with a signal-producing compound (e.g., an antibody labeled with an enzyme; a fluorescent compound; a chemiluminescent compound; a radioactive label; a direct visual label; etc.). For example, in a binding assay the test sample suspected of containing analyte can be mixed with a labeled anti-analyte antibody and incubated for the immunoreaction to occur. The reaction mixture is subsequently analyzed to detect either the label associated with an antibody/analyte complex (bound label) or the labeled antibody which is not complexed with analyte (free label), thereby enabling the detection or measurement of the analyte in the test sample.
Binding assays can be divided into two general categories known as homogeneous and heterogeneous assays. In the homogeneous assays, the signal produced by the bound label is different from the signal produced by the free label. As a result, bound and free label can be distinguished without physical separation of the individual reactants from the reaction mixture.
A well-known homogeneous binding assay is the enzyme-multiplied immunoassay technique (EMIT) which is disclosed in U.S. Pat. No. 3,817,837. In the EMIT assay, the analyte present in the patient's test sample and an enzyme-labeled analyte compete for a limited amount of anti-analyte antibody. The specific binding of the antibody to the analyte-enzyme conjugate modulates the conjugate's enzymatic activity such that the enzyme activity is proportional to the amount of analyte in the test sample. Homogeneous binding assays have the advantages of being rapid, easy to perform, and readily adapted to automation. Their disadvantages are that they are susceptible to interferences caused by non-analyte substances in the test sample, they are generally limited to assays for low molecular weight analytes, and they have a limited sensitivity.
In a heterogenous binding assay, the signal produced by the bound label is indistinguishable from the signal produced by the free label; therefore, the free label and the bound label must be separated from one another to distinguish between their respective signals. In some cases, the complex with which the bound label is associated will substantially differ in molecular weight from the free labeled reactant so that centrifugation can be used to separate the heavier complex.
An alternative to centrifugation involves attaching at least one of the binding assay's reactants to a solid support. The solid support can then be separated from the test sample and the remaining assay reagents to provide for the separation of the free and bound label. The separation of the solid support and reaction mixture can be accomplished either by drawing-off the remaining reaction mixture or by physically removing the solid phase from the reaction mixture. The solid support can also be treated or washed to remove interfering substances prior to the detection or measurement of the label associated with the solid phase.
Longer incubation times may be-required in the heterogeneous assay. This is because the kinetics of a reaction between a solid phase-bound specific binding member and its complementary binding partner tend to be slower than the kinetics of the same reaction when both binding members are in solution. The heterogeneous assays, however, are in general more sensitive than homogeneous assays and less prone to interferences, because interfering substances can be removed by the wash steps.
Variations to this general solid phase separation scheme have been developed, but they typically involve the binding of the analyte to a specific binding member which is attached to a solid phase. Generally, specific binding members are attached to or immobilized on the solid phase by adsorption or covalent bonding. Adsorption results from the action of the solid phase in attracting and holding the specific binding member. With covalent bonding, the specific binding member and the solid phase are chemically reacted to result in a bond which immobilizes the specific binding member on the solid phase.
The linkage between the solid phase and the immobilized specific binding member can greatly affect the binding of that specific binding member to the analyte. For example, antibodies have extremely specific structural, spatial and polar configurations which enable them to recognize and bind to a specific analyte (e.g., antigen). When antibodies are used in an assay for the detection of antigens, the antibodies may be the specific binding members linked to the solid phase. The proximity of the solid phase to the antibody, however, can partially or completely block the sites on the antibody to which the antigen binds. In addition, the linkage between the antibody and the solid phase can alter the conformation of the antibody and thereby affect the antibody's ability to bind to the analyte. The same limitations hold for the linkage of other specific binding members to a solid phase; the specific binding members may attach in a spectrum of positions ranging from complete steric hindrance of the binding site to unhindered access, and/or the conformation of the specific binding member can change upon linking to the solid phase so that its complementary binding partner can no longer recognize it or bind to it. As may be expected, the sensitivity of the assay declines with increasing levels of steric hindrance and loss of reactivity.
A conventional method for covalently attaching a proteinaceous specific binding member to a polymeric solid phase involves the use of carbodiimide to crosslink the amine groups of the protein to the carboxyl groups on the surface of the solid phase. Alternatively, gluteraldehyde is used to crosslink the amines of the protein to surface bound amines on the solid phase. These crosslinking methods, however, are poorly controlled, often resulting in protein/protein crosslinking and non-specific reactions, such as the over-modification of the protein which may result in a decrease in the binding capacity of the specific binding member. In addition, the protein that is so immobilized has poor reactivity, making it necessary to bind large amounts of the protein to the solid phase to obtain suitable assay sensitivity.
There have been two main approaches to solving the crosslinking problems associated with the heterogeneous assays. One approach has been to complete the reaction of the binding partners prior to the immobilization of the newly formed complex upon the solid phase. The other technique has been to extend the length of the linkage between the specific binding member and the solid phase. The linking or coupling agent must maintain the linkage during the chemical manipulations of the assay, as well as during the physical manipulations of washing and separation steps. Extended length heterobifunctional coupling agents have been described in co-owned and copending patent applications, U.S. Ser. No. 254,288, filed Oct. 11, 1988, now U.S. Pat. No. 5,002,883, and Ser. No. 114,930, filed Oct. 30, 1987, now abandoned, wherein the covalent attachment of specific binding members to the solid phase is accomplished using an extended length molecular chain having at least one linking group that is reactive with a chemical group on the solid phase and at least one linking group that is reactive with a chemical group on the specific binding member.